The "Thar Express" left Karachi with 200 passengers shortly before midnight before arriving in the Indian desert town of Munabao in the early hours of Saturday morning.
The train, named after the desert it traverses, connects Munabao in the western Indian state of Rajasthan with Khokrapar, in the Pakistani province of Sindh.
It is the second rail link between the two nuclear powers and marks another step forward in restoring confidence between them.
Pakistan will operate the two-way service every Saturday for the first six months of the year, and India will take over for second half of the year.
Pakistan and India agreed to reopen the rail link during a summit in New Delhi in April 2005.
A service between Lahore and New Delhi resumed in 2004, weeks after the two nations began a shaky peace process aimed at burying six decades of conflict.
The resumption of public transport, including new bus, rail and air links, are among the most popular measures of the peace dialogue as they are providing more opportunities for contact between the countries' two peoples.
They are enhancing business ties and uniting citizens separated decades ago when thousands of families on both sides were divided by the 1947 partition of the Indian subcontinent that created the new nation of Pakistan.
The two countries have fought three wars since the partition and gaining independence from Britain.
But their often-bitter relations, dominated by a territorial dispute and Islamic insurgency in Kashmir, have improved markedly since 2004.